TXNIP对糖尿病视网膜病大鼠ROS/RNS应激及DNA损伤的影响及机制分析

Effect of TXNIP on ROS/RNS stress and DNA damage in rats with diabetic retinopathy and its mechanism

目的观察硫氧还蛋白相互作用蛋白(TXNIP)对糖尿病视网膜病(DR)大鼠视网膜细胞凋亡的影响,并探讨其作用机制。方法将30只大鼠随机均分为三组:对照组、DM组和Azas组。DM组和Azas组用链脲佐菌素(STZ)诱发建立糖尿病模型,对照组大鼠注射同体积的枸橼酸缓冲液。Azas组大鼠给予玻璃体内注射10μmol/L的azaserine 2. 5μl,在建模成功后第23天和第27天各一次,均注射在右眼。对照组和DM组大鼠在相同时间点注射同体积的生理盐水。检测各组大鼠视网膜中己糖胺通路(HBP)活性和TXNIP的表达、活性氧(ROS)/活性氮(RNS)应激、线粒体功能、DNA损伤、DNA损伤修复和细胞凋亡等指标。结果与对照组相比,DM组中O-Glc NAc修饰、TXNIP表达、ROS含量和S-nitrosocysteine(SNO)表达显著增加(P <0. 05),Azas组中上述指标均显著低于DM组(P <0. 05)。与对照组相比,DM组中线粒体膜电位、复合物Ⅰ活性和复合物Ⅲ活性均显著降低(P <0. 05),Azas组中线粒体膜电位、复合物Ⅰ活性、复合物Ⅲ活性和复合物Ⅳ活性显著高于DM组(P <0. 05)。与对照组相比,DM组中彗星尾长、尾DNA含量百分比、尾距、Olive尾距、γH2AX和H3K9Me3的表达均显著增加(P <0. 05),Azas组中上述指标均显著低于DM组(P <0. 05)。与对照组相比,DM组中BRCA2的表达均显著降低(P <0. 05),Azas组中BRCA1、BRCA2、DNAPKcs、Ku70和Ku80的表达显著高于DM组(P <0. 05)。与对照组相比,DM组中细胞凋亡率和cleaved-caspase-3的表达显著增加(P <0. 05),Azas组中上述指标均显著高于DM组(P <0. 05)。结论 TXNIP抑制可缓解DM诱导的视网膜细胞凋亡,其机制可能与降低ROS/RNS应激和DNA损伤、增加线粒体功能和DNA损伤修复有关。

Objective To investigate the effect of TXNIP on retinal cell apoptosis in diabetic retinopathy( DR) rats,and to explore its mechanism. Methods 30 rats were randomly and averagely divided into three groups: control group,DM group( diabetes group) and Azas group( Azaserine,which inhibited TXNIP expression). The DM group and the Azas group were induced to establish a diabetes model by streptozotocin( STZ),and the control group rats were injected with the same volume of citrate buffer. The Azas group rats were given intravitreal injection of 10μmol/L of azaserine 2. 5 μl,once on the 23 rd and 27 th day after successful modeling into the right eye. The control group and the DM group rats were injected with the same volume of physiological saline at the same time point. The hexosamine channel( HBP) activity and TXNIP expression,ROS/RNS stress,mitochondrial function,DNA damage,DNA damage repair and apoptosis were detected in the retina of rats in each group. Results Compared with the control group,the levels of O-GlcNAc,TXNIP,ROS and SNO were significantly increased in the DM group( P <0. 05),and the above-mentioned indexes in the Azas group were significantly lower than that in the DM group( P < 0. 05). Compared with the control group,mitochondrial membrane potential and the activity of complex I and III were significantly decreased in DM group( P < 0. 05),mitochondrial membrane potential and the activity of complex I,III and IV in Azas group were significantly higher than those in the DM group( P <0. 05). Compared with the control group,the comet tail length,tail DNA content,tail moment,Olive tail moment,expressions of γH2 AX and H3 K9 Me3 in the DM group were significantly increased( P < 0. 05),and the above-mentioned indexes in the Azas group were significantly lower than those in the DM group( P < 0. 05). Compared with the control group,the expression of BRCA2 was significantly decreased in the DM group( P < 0. 05),and the expressions of BRCA1,BRCA2,DNA-PKcs,Ku70 and Ku80 in the Azas group were significantly higher than those in the DM group( P < 0. 05). Compared with the control group,the apoptotic rate and the expression of cleaved-caspase-3 in the DM group were significantly increased( P < 0. 05),and the above-mentioned indexes in the Azas group were significantly higher than those in the DM group( P < 0. 05). Conclusion TXNIP inhibition can alleviate DM-induced retinal cell apoptosis,and its mechanism may be related to the reduction of ROS/RNS stress and DNA damage,and the increase of mitochondrial function and DNA damage repair.